Previously we have studies the contribution of histone modifications, DNA methylation and their regulatory enzymes to transcriptional regulation in a variety of cellular systems. Recent studies have suggested cellular heterogeneity in gene expression even in the same cell population. The question is whether there is a similar heterogeneity in chromatin states in the apparently same cells. To address this question, we have worked on developing technologies that can be used to detect chromatin states in single-cells. DNase I hypersensitive sites (DHSs) provide important information on the presence of transcriptional regulatory elements and the states of chromatin in mammalian cells. Conventional DNase-Seq methods for genome-wide DHS profiling require millions of cells 4,5, which limits their utility. Here we report an ultrasensitive and robust strategy, called Pico-Seq, for detection of genome-wide DHSs in single cells. We show that DHS patterns at the single cell level are highly reproducible among individual cells, with the DHS detectability correlating well with gene expression levels and co-existence of multiple active histone modification marks at enhancers and promoters. Furthermore, the single-cell DHSs predict enhancers that regulate tissue-specific gene expression programs. Finally, we apply Pico-Seq to small numbers of tumor and adjacent normal cells, dissected from formalin-fixed paraffin-embedded (FFPE) tissue slides from thyroid cancer patients, and detect thousands of tumor-specific DHSs. Many of these DHSs are associated with promoters and potential enhancers of genes critically involved in cancer development and seem to have clinical relevance not previously appreciated. Analysis of the DHS sequences uncovers one single-nucleotide variant (chr18:52417839 G>C) in the tumor cells of a follicular thyroid carcinoma patient, which affects the binding site of the tumor suppressor protein p53 and correlates with decreased expression of its target gene TXNL1. In conclusion, Pico-seq can reliably detect DHSs in single cells, which greatly extends the range of applications of DHS analysis for both basic and translational research and may provide critical information for personalized medicine. In addition, we have worked with our collaborators using model systems including Drosophila flies and chicken to investigate the contribution of chromatin and epigenetic mechanisms to cellular memory and differentiation.